Voltage-activated potassium channels are membrane proteins that critically regulate electrical signaling in neurons and other excitable cells. Potassium channels arc particularly important for determining the tendency of excitable cells to produce repetitive signaling. Such repetitive activity is a central feature of electrical signaling in the nervous system and in the excitable tissues of the cardiovascular and endocrine systems. Normal repetitive signaling produces stimulus- frequency encoding, brain wave rhythms, and cardiac pacemaking; dysfunctions of rhythmic signaling lead to epilepsy, cardiac arrhythmia, and myotonia. The opening and closing of these potassium channels, which determines their effect on cellular electrical signaling, is accomplished by several separable but interrelated gating mechanisms. The outward rectifier K+ channels in this study have an activation gating process that opens the channel upon depolarization, and several distinguishable inactivation gating mechanisms. These inactivation mechanisms silence the channel after it has been opened, and incapacitate it for various lengths of time. Activity-induced inactivation gating can change the rhythmic signaling pattern of excitable cells. Two inactivation mechanisms, N-type and C-type, operate in K+ channels. Rapid N-type inactivation occurs by binding of a tethered blocker to the intracellular mouth of the channel. C-type inactivation, which is generally slower to occur and more long lasting, involves some conformational change at the extracellular mouth of the channel. This grant addresses the structural basis of these inactivation mechanisms and of their interaction. A general strategy is proposed to learn about the functional motions of the channel protein that correspond to gating. Genetically-altered channels will be made, with single cysteine residues substituted at specific locations. These introduced cysteines serve as targets for sulfhydryl-specific modifying reagents; their accessibility for modification can depend on the gating state of the channel. Previous work shows that C-type inactivation gating produces enormous changes in the chemical reactivity of a particular cysteine introduced at the outer mouth. Further work on this and neighboring sites should give a clear picture of the structural rearrangement in the outer mouth of the K+ channel that occurs with this form of inactivation. A similar approach will be applied to the conformational changes of the inner mouth that occur with activation gating. This work should provide fundamental new insights into channel gating that will help explain the physiological basis of repetitive signaling and its dysfunction. These insights will also increase the chances for rational development of drugs to control such disorders.